1. Field of the Invention
The present invention relates to a method for forming a proteoliposome which has a membrane protein incorporated in a membrane of a vesicle such as of a liposome, and particularly to a method for forming a proteoliposome in which a membrane protein is incorporated in a manner so as to control molecular orientation.
The present invention also relates to a method for preparing a giant proteoliposome having a diameter of 5 .mu.m or more in a simple process and in a large quantity under mild conditions.
The present invention further relates to a method for forming a giant proteoliposome having a membrane protein incorporated in a preferable molecular orientation.
2. Related Background Art
A liposome is an assembly of biologically functional molecules and is formed by dispersing and re-associating a lipid, which is a biomembrane-constituting molecule, in an aqueous solution and has a structure like a cellular membrane. The application of the liposome is under investigation as applied to medical diagnosis and treating medicines in medical care, and to production of useful materials in industrial fields.
The proteoliposomes which have a protein incorporated into the above mentioned liposomes can have a further higher function in addition to the function of the liposome because a functional molecule can be added thereto.
Proteoliposomes are prepared by various known methods corresponding to the uses thereof and the types of the protein. The known methods include a dialysis method, a sonication, a freeze-thaw method, etc.
In the dialysis method, a protein is solubilized together with a lipid by use of a detergent, and then the detergent is removed by dialysis to give small proteoliposomes having a diameter of 100 nm or less. In the sonication method, a protein is treated with a liposome by sonication, which gives small proteoliposomes having a small diameter of 100 nm or less. In the freeze-thaw method, a mixed solution of a protein and a liposome is frozen and thawed repeatedly to incorporate the protein into a lipid membrane, which gives a relatively large proteoliposome of several hundred nm in diameter.
On the other hand, there is a biomembrane comprising a membrane protein embedded with preferable molecular orientation. Such anisotropic membrane constitution realizes vectorial transfer of a substance or of information such as reception and conduction of excitation, and transportation of a substance. Hence, in applications of the proteoliposome to medical treatment and industry as well, a constant orientational incorporation of the membrane protein is advantageous to realize the function effectively.
Conventional methods for producing proteoliposomes are based on the process of chemical or physical dissolution or partial destruction of a lipid membrane, and subsequent reconstruction of a membrane with inclusion of a membrane protein. In the process, no means for positively controlling the molecular-orientation of the membrane protein is achieved.
Practically, in proteoliposome membranes prepared by conventional methods, the proteins are incorporated with random orientation.
Further, the diameter of the proteoliposomes depends on the respective method of preparation, and cannot be optionally selected.
Detailed studies on inclusion of membrane protein, especially a protein inherent in cellular membrane, into a lipid bilayer membrane in the formation of proteoliposome is found only in exceptional reports such as "Zakim: Biochemica et Biophysica Acta, 906, 33-68 (1987)" which discloses that a spontaneous insertion of a membrane protein solubilized by a detergent or a membrane protein free from both the detergent and membrane lipid into a lipid bilayer membrane may occur under appropriate conditions with activation energy lower than that of fusion between liposomes, regarding cytochrome, UDP glucuronosyltransferase, bacteriorhodopsin, etc. The inserted protein, however, is not preferably oriented, and is not orientationally controlled at all.
On the other hand, a giant proteoliposome having a diameter of 5 .mu.m or more is applicable as an artificial cell because it has a size equivalent to a cell and therefore can easily mimic various functions of the cell.
Its larger inner volume gives the advantages of a higher efficiency of substance retention, a higher efficiency of substance incorporation, etc., which allows development of high-performance microcapsules, and chemical sensors, etc. Further, the size exceeding 5 .mu.m in diameter thereof enables easy observation with an optical microscope and permits mechanical operation by means of micromanipulation or microinjection, which is hopefully adaptable to applications inherently different from the conventional proteoliposomes having smaller diameters.
Heretofore, known preparation methods of giant proteoliposomes have been limited to only a few methods such as an electric field fusion method and a giant liposome preparation method, none of which is practical.
The known methods for preparation of giant proteoliposomes, including an electric field fusion method, a stationary hydration method, a reverse phase evaporation method, and a freeze-thaw method are all under severe restriction, and are impractical except for restricted applications.
In the electric field fusion method, a suspension of small-diameter proteoliposomes prepared preliminarily in the conventional method is subjected to application of an electric field, leading to formation of giant proteoliposomes by aggregating and fusing the proteoliposomes. This method has the disadvantages that a low ionic concentration of the solution needs to be maintained because of the high electric field required, that only a small number of giant proteoliposomes can be prepared in one operation, and, further, that the diameter of proteoliposomes suitable for the fusion are limited and their preparation is not easy.
The stationary hydration method and the reverse phase evaporation method are both based on the application of a method for preparation of a giant liposome. The stationary hydration method which hydrates a thin lipid film formed on a glass wall under low ion strength to give a giant liposome, has serious disadvantages in that the ionic concentration of the solution should be 10.sup.-4 M or less and, further that the presence of a protein inhibits the formation of a proteoliposome. Thus this method is difficult for wide applications. The reverse phase evaporation method which employs an emulsion formed by mixing a solution containing a protein with an ether solution of a lipid tends to cause denaturation or deactivation of the protein, and thus incorporatable proteins are strictly limited.
The freeze-thaw method which has been employed for reconstruction of a membrane protein and is applicable to preparation of a proteoliposome having a diameter of 1 .mu.m or less, is known to be able to form only a small amount of giant proteoliposomes of 5-10 .mu.m in diameter, but is of no practical use because of the limited small number and small diameter of the particles.
As mentioned above, few methods are applicable in the preparation of giant proteoliposomes. All of the methods are strictly restricted and thus are not suitable for practical preparation. Accordingly, development of a method is desired which enables the preparation of a large amount of proteoliposomes by a simpler process under milder conditions.
Moreover, also for the giant proteoliposomes, a method is desired which allows the membrane protein to be incorporated into the membrane with the orientation of the membrane protein.